Many biological molecules (biomolecules) which are administered for therapeutic purposes are rapidly cleared from the circulation by glomerular filtration and, as a consequence, exhibit relatively short-lived pharmacological activity. Because of this rapid clearance, it is often necessary to administer larger amounts of the biomolecule at more frequent intervals in order to achieve the requisite therapeutic response. It has been shown that the clearance time of such therapeutic agents can, in many cases, be increased by the covalent attachment of water-soluble polymers. Examples of such water-soluble polymers are polyethylene glycol, copolymers of ethylene glycol and propylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone and polyproline [Abuchowski and Davis, Soluble Polymer-Enzyme Adducts. In: Enzymes as Drugs, Holcenberg and Roberts, eds., Wiley-Interscience, New York, N.Y., 1981, pp. 367-383; Newmark, et al., J. Appl. Biochem. 4:185-189 (1982); Katre, et al., Proc. Natl. Acad. Sci. USA 84:1487-1491 (1987)]. Conjugation with these polymers is claimed to increase the solubility of more hydrophobic drugs in water, eliminate aggregation of peptides and proteins and greatly reduce their immmunogenicity and antigenicity, and generally enhance the physical and chemical stability of therapeutic agents. Improved properties have been reported for proteins and peptides [Fuertges and Abuchowski, J. Controlled Release, 11:139-148 (1990)], low molecular weight therapeutics [Zalipsky et al, Eur. Polym. J., 19:1177-1183 (1983)] and, more recently, for liposomes [Woodle, et al., U.S. Pat. No. 5,013,556, issued 7th May 1991].
Despite numerous positive reports, a review of the scientific literature shows that covalent binding to a synthetic water soluble polymer does not predictably enhance the biological activity of biomolecules. For example, conjugation of streptokinase-plasmin complex and Pluronic F68, a block copolymer of ethylene and propylene glycol, destroys the activity of this enzyme [Newmark, et al., J. Appl. Biochem. 4:185-189, (1982)]. Dextran conjugates of several proteins have been reported to be strongly immunogenic in rabbits, sheep and guinea pigs [Richter et al., Int. Arch. Allergy, 42:885-902 (1972)]. The plasma half-lives of dextran conjugates of uricase in mice depend on the charge of the dextran; cationic and neutral dextran accelerate the elimination of the protein, while anionic dextran increases the half-life [Fujita et al., J. Controlled Release, 11:149-156 (1990)]. Polyvinylpyrrolidone conjugates have been found to be immunogenic [Abuchowski and Davis, Soluble Polymer-Enzyme Adducts. In: Enzymes as Drugs, Holcenberg and Roberts, eds., Wiley-Interscience, New York, N.Y., 1981, pp. 367-383], and to destroy the biological activity of ribonuclease (Veronese, et al, J. Bioactive Comp. Polym., 5:167-178 (1990)). In contrast , ribonuclease retains its activity after conjugation with polyethylene glycol (PEG) (Veronese, et al., Appl. Biochem. Biotech., 11:141-152 (1985)]. Even the degree of substitution and type of conjugate linkage can impact on the biological activity of the biomolecule. For example, European patent application no. 0,335,423 A2, published on Apr. 10, 1989, discloses that progressive conjugation of cyanuric-chloride activated PEG to the protein granulocyte colony stimulating factor (G-CSF) proportionately reduces its biological activity in vitro. These examples teach that conjugation of biomolecules with a water soluble polymer is not a predictable means of improving the biological properties of therapeutic agents.
Poly-1,3-dioxolane is a synthetic water soluble polymer comprised of the formula --[--O--CH.sub.2 --O--CH.sub.2 --CH.sub.2 --].sub.n --. Poly-1,3,6-trioxocane, is a structurally related polymer with the formula --[--O--CH.sub.2 --O--CH.sub.2 CH.sub.2 --O--CH.sub.2 CH.sub.2 --].sub.n --. Here, n is the number of repeating mer units which can theoretically range from less than ten to many thousands. Both polymers contain the oxymethylene-oxyethylene part structure. Although polymers with this structure are known in the art [Franta, et al., Die Makromol. Chem. 191:1689-1698 (1990); Velichova, et al, J. Polymer Science, Part A, 28:3145-54 (1990)], none has heretofore been combined with biomolecules to enhance the activity of the latter.
A simple method of preparation of polymers containing the oxymethylene-oxyethylene part structure is ring-opening polymerization of the cyclic monomers 1,3-dioxolane or 1,3,6-trioxocane. The ratios of the methyleneoxy to ethyleneoxy groups in the resulting polymers, poly-1,3-dioxolane and poly-1,3,6-trioxocane, are 1:1 and 1:2, respectively. This ratio can be changed to a wide range of non-integer values by copolymerization of 1,3-dioxolane and 1,3,6-trioxocane with each other or with other monomers. For example, 1,3,5-trioxane, which has the formula (CH.sub.2 O).sub.3, has been copolymerized with either 1,3-dioxolane or 1,3,6-trioxocane to increase the methyleneoxy content of the polymer. Alternatively, copolymerization with oxirane may be used to increase the ethyleneoxy content. The conjugates of these oxymethylene-oxyethylene polymers with biological agents constitutes the present invention.
None of the previously described methods for preparation of polymer-conjugates adducts have disclosed details of how to modify a biomolecule with the subject polymers so that its biological activity in vivo is enhanced.